Macrophages are the cornerstone of the innate immune response to infection. They define the nature of pathogens through germ-line encoded pattern recognition receptors that then process the information and specify appropriate responses. The Toll-like receptors are prototypic pattern recognition receptors; TLR4 recognizes the Gram-negative cell-wall component LPS, while TLR5 detects bacterial flagellin. TLR4 and TLR5 trigger both common and distinct signaling pathways in macrophages, and these elicit different responses to different pathogens. To obtain the structure of these pathways we will use isotope-coded affinity tags (ICAT) to quantify early phosphorylation events triggered by TLR4 and TLR5, and correlate these events with specific biological outcomes defined by cDNA arrays representing more than 1600 macrophage inflammatory genes. These data will permit development of a predictive model that will be confirmed and refined by additional perturbations of constriction points in the signaling pathways. This model will be a starting point for the development of a comprehensive network model of macrophage signaling. This approach will ultimately be applicable to a broad array of biological systems.